Loss of microenvironmental Ptn leads to enhanced stem cell maintenance in vivo. (A) Experimental design of serial transplantation experiments. WT Ly5.1 BM cells (2 × 10⁵) were transplanted into Ptn^−/− and Ptn^+/+ littermate (Ly-5.2⁺) recipients irradiated with 9 Gy. Sixteen weeks after transplantation, recipients were killed, and 1 × 10⁶ BM cells were serially transplanted into WT secondary recipients. Again, mice were killed 16 weeks after transplantation, and 2 × 10⁶ BM cells were serially transplanted into tertiary irradiated WT mice. Finally, 5 × 10⁶ BM cells of these tertiary recipients were again transplanted into irradiated quaternary WT recipients. (B) Engraftment levels as percentage of Ly-5.1⁺ donor cells in BM of first-degree recipients, n = 5 (+/+), n = 10 (−/−). (C) Primary transplantation, engraftment levels in PB 16 weeks after transplantation. (D) Secondary transplantation, engraftment levels in BM 16 weeks after transplantation, percentage of donor cells in total cell count of 4 long bones, n = 10 (Ptn^+/+), n = 8 (Ptn^−/−). (E) Secondary transplantation, engraftment levels in PB 16 weeks after transplantation. (F) Tertiary transplantation, engraftment levels in BM 16 weeks after transplantation as percentage of donor cells; n = 7 (Ptn^+/+), n = 5 (Ptn^−/−). (G) Tertiary transplantation, engraftment levels in PB 16 weeks after transplantation. (H) Quaternary transplantation, engraftment levels in BM 16 weeks after transplantation, as percentage of donor cells; n = 5 (Ptn^+/+), n = 6 (Ptn^−/−). (I) Quaternary transplantation, engraftment levels in PB 16 weeks after transplantation. (J) Calculated absolute numbers of Cd34⁻LSK and MMP cells per 4 long bones in the serial transplantations, based on the number of transplanted BM cells. (K) Flow cytometric analysis of BM of quaternary transplants. LSK and MMP are gated in Lin⁻ cells, Cd34⁻LSKs were gated in the LSK cells. All values are mean ± SEM; *P < .05.